ADP-RIBOSYLATION FACTOR 1-REGULATED PHOSPHOLIPASE-D ACTIVITY IS LOCALIZED AT THE PLASMA-MEMBRANE AND INTRACELLULAR ORGANELLES IN HL60 CELLS

ADP-ribosylation factor (ARF), a small GTPase required for vesicle for mation, has been identified as an activator of phospholipase D (PLD), thus implying that PLD is localized at intracellular organelles. HL60 cells were prelabelled with [C-14]acetate for 72 h and, after disrupti on, fractionated on a linear sucrose gradient. ARF1-regulated PLD acti vity in each fraction was assessed by measurement of phosphatidylethan ol production. Two peaks of activity were identified, coincident with markers for Golgi/endoplasmic reticulum/granules (endomembranes) and p lasma membrane respectively. Analysis of the fractions using exogenous phosphatidylcholine as substrate confirmed the presence of ARF1-depen dent PLD activity in endomembranes and plasma membrane, and also ident ified an additional activity in the cytosol. In formyl-Met-Leu-Phe-sti mulated cells, PLD activity as assessed by phosphatidylethanol formati on was also associated with both the plasma membrane and endomembranes . Since ARF1-regulated PLD activity requires phosphatidylinositol 4,5- bisphosphate (PIP2), the distributions of inositol lipids and the kina ses responsible for lipid phosphorylation were examined. PIP2 was high ly enriched at the plasma membrane, whereas phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PI4P), the precursors for PIP2 s ynthesis, were found predominantly at endomembranes. The distribution of PI 4-kinase and PI4P 5-kinase activities confirmed the plasma membr ane as the major site of PIP2 production. However, endomembranes posse ssed substantial PI 4-kinase activity and some PI4P 5-kinase activity, illustrating the potential for PIP2 synthesis. It is concluded that: (1) ARF1-regulated PLD activity is localized at endomembranes and the plasma membrane, (2) PIP2 is available at both membrane compartments t o function as a cofactor for ARF-regulated PLD, and (3) in intact cell s, formyl-Met-Leu-Phe stimulates PLD activity at endomembranes as well as plasma membrane.